Efficient biosynthesis of cinnamyl alcohol by engineered Escherichia coli overexpressing carboxylic acid reductase in a biphasic system

Background: Cinnamyl alcohol is not only a kind of flavoring agent and fragrance, but also a versatile chemical applied in the production of various compounds. At present, the preparation of cinnamyl alcohol depends on plant extraction and chemical synthesis, which have several drawbacks, including limited scalability, productivity and environmental impact. It is therefore necessary to develop an efficient, green and sustainable biosynthesis method. Results: Herein, we constructed a recombinantEscherichia coliBLCS coexpressing carboxylic acid reductase fromNocardia iowensisand phosphopantetheine transferase fromBacillus subtilis. The strain could convert cinnamic acid into cinnamyl alcohol without overexpressing alcohol dehydrogenase or aldo-keto reductase. Severe product inhibition was found to be the key limiting factor for cinnamyl alcohol biosynthesis. Thus, a biphasic system was proposed to overcome the inhibition of cinnamyl alcohol via in situ product removal. With the use of a dibutyl phthalate/water biphasic system, not only was product inhibition removed, but also the simultaneous separation and concentration of cinnamyl alcohol was achieved. Up to 17.4 mM cinnamic acid in the aqueous phase was totally reduced to cinnamyl alcohol with a yield of 88.2%, and the synthesized cinnamyl alcohol was concentrated to 37.4 mM in the organic phase. This process also demonstrated robust performance when it was integrated with the production of cinnamic acid froml-phenylalanine. Conclusion: We developed an efficient one-pot two-step biosynthesis system for cinnamyl alcohol production, which opens up possibilities for the practical biosynthesis of natural cinnamyl alcohol at an industrial scale.